1. Field of the Invention
The present invention relates to modified stem cells. The present invention also relates to methods of modifying stem cells for the purpose of stem cell expansion in-vitro. The invention also further relates to methods of enhancing stem cell proliferation and self-renewal in-vivo. The invention further relates to regeneration of tissues by using the modified stem cells.
2. General Background and State of the Art
Stem cell therapy is a new approach for medical intervention for many intractable diseases. Two types of stem cells can be used for regeneration of tissues, adult-type stem cells and embryonic stem cells. Adult-type stem cells including cord blood stem cells, bone marrow-derived stem cells, pancreatic stem cells or hepatic stem cells are very limited in the numbers that can be obtained from given tissues.
The pluripotent adult-type stem cells have the capacity to self-renew themselves and undergo multi-lineage differentiation. However, molecular mechanisms regulating such self-renewal and expansion of adult-type stem cells, in particular, hematopoietic stem cells has been illuminating.
Hematopoietic stem cells are one type of adult stem cells best characterized for heterogeneity in pluripotency and ability to proliferate in-vivo and in-vitro.
The most primitive undifferentiated state of hematopoietic stem cells have capacity to self-renew themselves and give rise to multi-lineage long term repopulation after transplantation into irradiated recipients. These cells are identified and defined in the transplantation model as CRU (competitive repopulating unit) (Larochelle 1996)
Another class of progenitor cells involved in hematopoiesis is assessed by their ability to form spleen colony (CFU-S: colony forming unit-spleen), which has more differentiated phenotypes and short-term repopulating ability.
One limiting factor in optimizing stem cell therapy using these pluripotent stem cells is that they are very prone to differentiation and loss of stem cell properties, leading to net loss of stem cell numbers after manipulation. Strategy to expand these stem cells including cytokine-aided ex-vivo culture, or modification of genes that are involved in regulation of stem cell proliferation has been tried. However, ex-vivo culture tends to give rise to more differentiated phenotype of stem cells despite the net increase of total cell numbers (Danet 2001, Dorrel 2000, Xu 2001).
To circumvent these limitations, several approaches were made by applying genetic modification in the stem cells including growth factor receptors or transcription factors with variable degree of increase in stem cell activities achieved (Hanazono 2002, Sauvageau 1995).
Recent studies in stem cell differentiation has shown that adult stem cells have the ability to differentiate into unexpected tissue types as well as expected tissue types. For example, hematopoietic stem cells can differentiate into neuronal cells, liver cells, renal cells, heart muscle cells, vascular tissues as well as hematopoietic lympho-myeloid lineages (Eglitis 1997, Poulsome 2001, Lagasse 2000, Orlic 2000).
STAT3 is a signal transducing molecule triggered by activation of IL-6 family growth factors, and gp-130 receptor family. The molecule is composed of DNA binding domain near N-terminus, SH2 domain, and transactivation domain near C-terminus.
Upon receiving signal from gp-130 receptors, JAK2 kinase is activated, which then phosphorylates tyrosine residue of STAT3. STAT3 then undergoes dimerization and nuclear localization for further activation of target genes.
Recently, it was shown that STAT3 activity can be constitutively activated by substituting several amino acid residue with cysteine residues (named STAT3-C), which leads to formation of disulfide bridge to dimerize this molecule in the absence of tyrosine phosphorylation or serine phosphorylation. (Bromberg 1999)
It was shown that functional knock out of STAT3 genes leads to loss of self-renewal and differentiation in the embryonic stem cells, and that STAT3 function is required for maintenance of undifferentiated phenotype of embryonic stem cells. (Matsuda 1999, Niwa 1998)
However, it is less likely that molecular mechanisms regulating adult hematopoietic stem cells are similarly regulated as embryonic stem cells, since transplantation of embryonic stem cells does not give rise to new reconstitution of bone marrows, and those mechanisms for adult hematopoietic stem cells has been illusive.
Recently, it was reported that over expression of dominant negative form of STAT3 can suppress bone marrow repopulation by such genetically modified hematopoietic stem cells, thus first identifying STAT3 activation as a mechanism that is necessary for in-vivo repopulation of transplanted stem cells, which is unique to adult hematopoietic stem cells (Oh, 2002).
However, in this study, over expression of wild type STAT3 genes did not affect any stem cell activity for bone marrow reconstitution. Thus, the reference did not disclose or suggest stem cell expansion by genetic manipulation of STAT3.
U.S. Pat. No. 6,235,873 discloses a mutant of STAT3 (STAT3-C) to increase dimerization of STAT3 protein in cells. STAT3-C contains two cysteine residues in the C-terminus of the protein in SH2 domain. However, the '873 patent does not disclose or suggest stem cells and related progenitor cells and their functional modulation of regenerative activities.
Matsuda et al. EMBO Journal, 18, 15, 4261-4269 (1999) discloses that STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. Niwa et al., Genes & Development, 12, 13, 2048-2060 (1998) discloses that self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. These references describe the role of STAT3 for maintaining undifferentiated state of embryonic stem cell. A dominant negative form of STAT3 (STAT3-F) and inducible form of STAT3 (STAT3-ER) was used to demonstrate the requirement of STAT3 activity in maintaining the undifferentiated phenotype of embryonic stem cells. However, these references fail to disclose or suggest that activated form of STAT3 enhance the STAT3 activities above the unmanipulated state. In addition, these references fail to disclose or suggest usage of adult stem cells or primary stem cells such as hematopoietic stem cells, nor their increased activities during in-vivo regeneration of tissues.
Oh et al., Oncogene July 18;21(31):4778-87 (2002) discloses that overexpression of dominant negative form of STAT3 suppresses the repopulating activity of hematopoietic stem cells. However, overexpression of wild-type STAT3 does not exert any effect on these stem cells. Thus, the reference fails to disclose or suggest that stem cell activity is enhanced by activated form of STAT3.
The present application describes exogenous expression of activated form of STAT3 (exemplified with STAT3-C) leads to net expansion of stem cells with increase in their self-renewal and regenerative capacity.